Some insect immune responses require activation of plasma proteins by proteases in hemolymph. These include proteolytic processing of cytokine precursors, including the Toll ligand Spatzle and the plasmatocyte spreading peptide. Another protease-mediated response is the activation of phenoloxidase zymogens, which then oxidize circulating diphenols to quinones, precursors for deposition of melanin on the surface of invading organisms. Such innate immune responses are likely to affect the outcome of infections of insect vectors with the pathogens and parasites they transmit. Responses modulated by proteases in blood are not simply linear pathways, but instead each protease may cleave multiple substrates, and active proteases may be regulated by several inhibitors. Previous work from our laboratory and others has established the lepidopteran insect, Manduca sexta, as a model system well suited for biochemical investigation of hemolymph proteases. The new availability of a M. sexta genome sequence make possible the investigation of serine protease webs in M. sexta hemolymph, using biochemical systems approaches. This research will be aimed at understanding hemolymph protease pathways and their regulation. The proteomics approach may reveal previously undiscovered protease-activated immune molecules of insects. The specific aims of the project are to: 1. Characterization of the plasma protein degradome to identify substrates for clip domain proteases and study their immune functions. 2. Continue to study the regulation of protease pathways in hemolymph by serpins. 3. Investigate the activation of the pathway-initiating modular protease, HP14. The long term goals of the research are to gain a thorough understanding of the regulation of protease cascades that mediate innate immune responses in Manduca, to apply this knowledge to insect vectors of human diseases, and to apply advantages of the Manduca system for fundamental studies on the regulation of serine protease activity.